The Preparation And The Effect Of Proliferation For HepG 2 Cells Of Specific Selenium-binding Peptide In Grifola Frondosa

In this thesis,the protein of Grifola frondosa was hydrolyzed by alkaline.The chelation technology of Grifola frondosa protein hydrolysate(GFPH)with selenium was optimized with response surface methodology;the ultrafiltration,gel chromatography and reversed-phase high performance liquid chromatography methods was used to obtain the peptide with high selenium-binding capacity;the selenium-binding mechanism was investigated with a combined methods including infrared spectrum,UV spectrum and 1HNMR;The antitumor activity of Grifola frondosa protein hydrolysate-selenium was investigated by establishing the HepG 2 cell,and the RT-qPCR was conducted to investigate the metabolic factors and pathways.1.The response surface method was used to investigate the optimized chelation technology of Grifola frondosa protein hydrolysate with selenium.The results showed that the optimum process parameters for the Grifola frondosa protein hydrolysate-selenium chelation were the ratio of hydrolysates to sodium selenite(v:v)of 6:4,reaction time of 90 min,pH of 9.0 and reaction temperature of 45℃,the selenium binding capacity was 2979.45±8.77 μg/g which was close to predictive value and applied to subsequent experiment.2.To isolate of peptides high selenium-binding capacity,the ultrafiltration,gel chromatography and reversed-phase high performance liquid chromatography methods was used.Five specific selenium-binding peptides were obtained through time-of-flight mass spectrometer,respectively obtained were SL(219 Da),TL(233 Da),VL(231 Da),ML(263 Da)and RL((359 Da).The highest selenium-binding capacity of tripeptide Arg-Leu-Ala(RLA)was 84.47±1.21 mg/g.And compared to the peptides before purification,the selenium-binding capacity was improved by more than three times.3.The physicochemical properties and structural characteristics of RLA-Se chelate were researched.The scanning electron microscope and XRD analysis proved that RLA-Se chelate was different from RLA and became a new compound.And the results of 1H NMR spectrum showed that after binding with selenium ion,the structure of RLA-Se chelate was more condensed with polymerization.Compared to UV spectrum of RLA,RLA-Se chelate had a hyperchromic effect at maximum absorption peak as-CH3 in the side-chain folding with π system.In conclusion,the amidogen and carbonyl participated in binding with selenium ion with the help of methyl residues.4.The Karber’s method method was employed to evaluate the anti-proliferation effect of RLA-Se chelate on HepG 2 cell models.The IC50 value was 286.42 μg/mL.The cell morphology showed that HepG 2 cell treated with RLA-Se chelate increased the mount of shrunken cells and lostd the adherence character.The research of cell cycle distribution and apoptotic rate were also detected and the results showed that apoptosis was the reason for the proliferation inhibition.To investigate metabolic factor and pathways,the RT-qPCR was conducted.And the research demonstrated that apoptosis related gene,like p53 gene and the death receptor pathway were related to inhibit HepG 2 cell proliferation.